Diana Seidel

Induced Tauopathy in a Novel 3D-Culture Model Mediates Neurodegenerative Processes: A Real-Time Study on Biochips

Tauopathies including Alzheimer’s disease represent one of the major health problems of aging population worldwide. Therefore, a better understanding of tau-dependent pathologies and consequently, tau-related intervention strategies is highly demanded. In recent years, several tau-focused therapies have been proposed with the aim to stop disease progression. However, to develop efficient active pharmaceutical ingredients for the broad treatment of Alzheimer’s disease patients, further improvements are necessary for understanding the detailed neurodegenerative processes as well as the mechanism and side effects of potential active pharmaceutical ingredients (API) in the neuronal system. In this context, there is a lack of suitable complex in vitro cell culture models recapitulating major aspects of taupathological degenerative processes in sufficient time and reproducible manner. Herewith, we describe a novel 3D SH-SY5Y cell-based, tauopathy model that shows advanced characteristics of matured neurons in comparison to monolayer cultures without the need of artificial differentiation promoting agents. Moreover, the recombinant expression of a novel highly pathologic fourfold mutated human tau variant lead to a fast and emphasized degeneration of neuritic processes. The neurodegenerative effects could be analyzed in real time and with high sensitivity using our unique microcavity array-based impedance spectroscopy measurement system. We were able to quantify a time- and concentration-dependent relative impedance decrease when Alzheimer’s disease-like tau pathology was induced in the neuronal 3D cell culture model. In combination with the collected optical information, the degenerative processes within each 3D-culture could be monitored and analyzed. More strikingly, tau-specific regenerative effects caused by tau-focused active pharmaceutical ingredients could be quantitatively monitored by impedance spectroscopy. Bringing together our novel complex 3D cell culture taupathology model and our microcavity array-based impedimetric measurement system, we provide a powerful tool for the label-free investigation of tau-related pathology processes as well as the high content analysis of potential active pharmaceutical ingredient candidates.

Impedimetric real-time monitoring of neural pluripotent stem cell differentiation process on microelectrode arrays

In todays neurodevelopment and -disease research, human neural stem/progenitor cell-derived networks represent the sole accessible in vitro model possessing a primary phenotype. However, cultivation and moreover, differentiation as well as maturation of human neural stem/progenitor cells are very complex and time-consuming processes. Therefore, techniques for the sensitive non-invasive, real-time monitoring of neuronal differentiation and maturation are highly demanded. Using impedance spectroscopy, the differentiation of several human neural stem/progenitor cell lines was analyzed in detail. After development of an optimum microelectrode array for reliable and sensitive long-term monitoring, distinct cell-dependent impedimetric parameters that could specifically be associated with the progress and quality of neuronal differentiation were identified. Cellular impedance changes correlated well with the temporal regulation of biomolecular progenitor versus mature neural marker expression as well as cellular structure changes accompanying neuronal differentiation. More strikingly, the capability of the impedimetric differentiation monitoring system for the use as a screening tool was demonstrated by applying compounds that are known to promote neuronal differentiation such as the γ-secretase inhibitor DAPT. The non-invasive impedance spectroscopy-based measurement system can be used for sensitive and quantitative monitoring of neuronal differentiation processes. Therefore, this technique could be a very useful tool for quality control of neuronal differentiation and moreover, for neurogenic compound identification and industrial high-content screening demands in the field of safety assessment as well as drug development.

A novel 3D label-free monitoring system of hES-derived cardiomyocyte clusters: a step forward to in vitro cardiotoxicity testing.

Unexpected adverse effects on the cardiovascular system remain a major challenge in the development of novel active pharmaceutical ingredients (API). To overcome the current limitations of animal-based in vitro and in vivo test systems, stem cell derived human cardiomyocyte clusters (hCMC) offer the opportunity for highly predictable pre-clinical testing. The three-dimensional structure of hCMC appears more representative of tissue milieu than traditional monolayer cell culture. However, there is a lack of long-term, real time monitoring systems for tissue-like cardiac material. To address this issue, we have developed a microcavity array (MCA)-based label-free monitoring system that eliminates the need for critical hCMC adhesion and outgrowth steps. In contrast, feasible field potential derived action potential recording is possible immediately after positioning within the microcavity. Moreover, this approach allows extended observation of adverse effects on hCMC. For the first time, we describe herein the monitoring of hCMC over 35 days while preserving the hCMC structure and electrophysiological characteristics. Furthermore, we demonstrated the sensitive detection and quantification of adverse API effects using E4031, doxorubicin, and noradrenaline directly on unaltered 3D cultures. The MCA system provides multi-parameter analysis capabilities incorporating field potential recording, impedance spectroscopy, and optical read-outs on individual clusters giving a comprehensive insight into induced cellular alterations within a complex cardiac culture over days or even weeks.

Functional bioassays for immune monitoring of high-risk neuroblastoma patients treated with ch14.18/CHO anti-GD2 antibody.

Effective treatment of high-risk neuroblastoma (NB) remains a major challenge in pediatric oncology. Human/mouse chimeric monoclonal anti-GD2 antibody (mAb) ch14.18 is emerging as a treatment option to improve outcome. After establishing a production process in Chinese hamster ovary (CHO) cells, ch14.18/CHO was made available in Europe for clinical trials. Here, we describe validated functional bioassays for the purpose of immune monitoring of these trials and demonstrate GD2-specific immune effector functions of ch14.18/CHO in treated patients. Two calcein-based bioassays for complement-dependent- (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were set up based on patient serum and immune cells tested against NB cells. For this purpose, we identified LA-N-1 NB cells as best suited within a panel of cell lines. Assay conditions were first established using serum and cells of healthy donors. We found an effector-to-target (E:T) cell ratio of 20∶1 for PBMC preparations as best suited for GD2-specific ADCC analysis. A simplified method of effector cell preparation by lysis of erythrocytes was evaluated revealing equivalent results at an E:T ratio of 40∶1. Optimal results for CDC were found with a serum dilution at 1∶8. For validation, both within-assay and inter-assay precision were determined and coefficients of variation (CV) were below 20%. Sample quality following storage at room temperature (RT) showed that sodium-heparin-anticoagulated blood and serum are stable for 48 h and 96 h, respectively. Application of these bioassays to blood samples of three selected high-risk NB patients treated with ch14.18/CHO (100 mg/m2) revealed GD2-specific increases in CDC (4.5–9.4 fold) and ADCC (4.6–6.0 fold) on day 8 compared to baseline, indicating assay applicability for the monitoring of multicenter clinical trials requiring sample shipment at RT for central lab analysis.

Targeting of MYCN by means of DNA vaccination is effective against neuroblastoma in mice

The MYCN oncogene is a strong genetic marker associated with poor prognosis in neuroblastoma (NB). Therefore, MYCN gene amplification and subsequent overexpression provide a possible target for new treatment approaches in NB. We first identified an inverse correlation of MYCN expression with CD45 mRNA in 101 NB tumor samples. KEGG mapping further revealed that MYCN expression was associated with immune-suppressive pathways characterized by a down-regulation of T cell activation and up-regulation of T cell inhibitory gene transcripts. We then aimed to investigate whether DNA vaccination against MYCN is effective to induce an antigen-specific and T cell-mediated immune response. For this purpose, we generated a MYCN-expressing syngeneic mouse model by MYCN gene transfer to NXS2 cells. MYCN-DNA vaccines were engineered based on the pCMV-F3Ub plasmid backbone to drive ubiquitinated full-length MYCN-cDNA and minigene expression. Vaccines were delivered orally with attenuated S. typhimurium strain SL7207 as a carrier. Immunization with both MYCN-DNA vaccines significantly reduced primary tumor growth of MYCN-expressing NB cells in contrast to negative controls. The immune response was mediated by tumor-infiltrating T cells in vivo, which revealed MYCN-specific and MHC class I-restricted lysis of inducible MYCN-expressing NB target cells in vitro. Finally, these antigen-specific T cells also killed MYCN-negative mammary carcinoma cells pulsed with MYCN peptides in contrast to controls. In summary, we demonstrate proof of concept that MYCN can be targeted by DNA vaccination, which may provide an approach to overcoming MYCN immune-suppressive activities in patients with MYCN-amplified disease.

Validated detection of human anti-chimeric immune responses in serum of neuroblastoma patients treated with ch14.18/CHO.

Human/mouse chimeric monoclonal antibody (mAb) ch14.18/CHO is directed against disialoganglioside GD2. Activity and efficacy of this mAb are currently determined in ongoing clinical Phase II and -III studies in high-risk neuroblastoma (NB). Based on the chimeric nature of this mAb, some patients may develop a human anti-chimeric immune response (Mirick et al., 2004) which impacts on pharmacokinetics and may induce anti-anti-idiotype (Id) mAb with a potential survival benefit. Therefore, a validated method of quantitative detection of human anti-chimeric antibodies (HACA) in serum samples of NB patients treated with ch14.18/CHO is an important tool for monitoring of clinical trials. Here, we report a validated sandwich enzyme-linked immunosorbent assay (ELISA) according to the one arm binding principle using ch14.18/CHO as a capture mAb and biotinylated ch14.18/CHO mAb for detection. Ganglidiomab, a monoclonal anti-Id Ab to ch14.18/CHO (Lode et al., 2013), was used as a standard for assay validation and HACA quantification. Systematic evaluation of the established ELISA procedure revealed an optimal serum sample dilution factor of 1:160. Assay validation was accomplished with a set of tailored quality controls (QC) containing distinct concentrations of ganglidiomab (3 and 15μg/ml). The coefficients of variation (CV) for all within-assay and inter-assay measurements using QCs were under 20% and the limit of detection (LOD) was 1.1μg/ml. Three patients (P1, P2, P3) treated with a 10day continuous infusion of 100mg/m(2) of ch14.18/CHO were selected for analysis with this assay. Selection was based on ch14.18/CHO drug level on day 8 in cycle 2 of >10μg/ml (expected) (P1) and of <2μg/ml (unexpected) (P2 and P3). Both patients with unexpected low ch14.18/CHO levels revealed a strong signal in the HACA ELISA. Interestingly, ch14.18/CHO-mediated complement-dependent cytotoxicity (CDC) could not be detected in P2 in contrast to P3 suggesting anti-NB activity even in the presence of HACA. We showed that neither eight freeze-thaw cycles nor storage at room temperature for up to 168h affected HACA stability in serum. In summary, we describe a validated ELISA method suitable for the assessment of HACA in NB patients treated with ch14.18/CHO.

Generation and Characterization of a Human/Mouse Chimeric GD2-Mimicking Anti-Idiotype Antibody Ganglidiximab for Active Immunotherapy against Neuroblastoma.

Vaccination with proteins mimicking GD2 that is highly expressed on neuroblastoma (NB) cells is a promising strategy in treatment of NB, a pediatric malignancy with poor prognosis. We previously showed efficacy of ganglidiomab in vivo, a murine anti-idiotype (anti-Id) IgG1. In order to tailor immune responses to variable regions, we generated a new human/mouse chimeric anti-Id antibody (Ab) ganglidiximab by replacing murine constant fragments with corresponding human IgG1 regions. DNA sequences encoding for variable regions of heavy (VH) and light chains (VL) were synthesized by RT-PCR from total RNA of ganglidiomab-producing hybridoma cells and further ligated into mammalian expression plasmids with coding sequences for constant regions of human IgG1 heavy and light chains, respectively. We established a stable production cell line using Chinese hamster ovarian (CHO) cells co-transfected with two expression plasmids driving the expression of either ganglidiximab heavy or light chain. After purification from supernatants, anti-idiotypic characteristics of ganglidiximab were demonstrated. Binding of ganglidiximab to anti-GD2 Abs of the 14.18 family as well as to NK-92tr cells expressing a GD2-specific chimeric antigen receptor (scFv(ch14.18)-zeta) was shown using standard ELISA and flow cytometry analysis, respectively. Ganglidiximab binding affinities to anti-GD2 Abs were further determined by surface plasmon resonance technique. Moreover, binding of anti-GD2 Abs to the nominal antigen GD2 as well as GD2-specific Ab-mediated cytotoxicity (ADCC, CDC) was competitively inhibited by ganglidiximab. Finally, ganglidiximab was successfully used as a protein vaccine in vivo to induce a GD2-specific humoral immune response. In summary, we report generation and characterization of a new human/mouse chimeric anti-Id Ab ganglidiximab for active immunotherapy against NB. This Ab may be useful to tailor immune responses to the paratope regions mimicking GD2 overexpressed in NB.

A novel 384-multiwell microelectrode array for the impedimetric monitoring of Tau protein induced neurodegenerative processes

Over the last decades, countless bioelectronic monitoring systems were developed for the analysis of cells as well as complex tissues. Most studies addressed the sensitivity and specificity of the bioelectronic detection method in comparison to classical molecular biological assays. In contrast, the up scaling as a prerequisite for the practical application of these novel bioelectronic monitoring systems is mostly only discussed theoretically. In this context, we developed a novel 384-multiwell microelectrode array (MMEA) based measurement system for the sensitive label-free real-time monitoring of neurodegenerative processes by impedance spectroscopy. With respect to the needs of productive screening systems for robust and reproducible measurements on high numbers of plates, we focused on reducing the critical contacting of more than 400 electrodes for a 384-MMEA. Therefore, we introduced an on top array of immersive counter electrodes that are individually addressed by a multiplexer and connected all measurement electrodes on the 384-MMEA to a single contact point. More strikingly, our novel approach provided a comparable signal stability and sensitivity similar to an array with integrated counter electrodes. Next, we optimized a SH-SY5Y cell based tauopathy model by introducing a novel 5-fold Tau mutation eliminating the need of artificial tauopathy induction. In combination with our novel 384-MMEA based measurement system, the concentration and time dependent neuroregenerative effect of the kinase inhibitor SRN-003-556 could be quantitatively monitored. Thus, our novel screening system could be a useful tool to identify and develop potential novel therapeutics in the field of Tau-related neurodegenerative diseases.

Abstract A032: Immunotherapy with ch14.18/CHO in combination with IL2 is active and effective in high-risk relapsed/refractory neuroblastoma patients

Background: Ganglioside GD 2 is a glycolipid highly expressed on neuro-ectodermal tumors including melanoma and neuroblastoma (NB). It is ranked on position 12 of the list of tumor associated antigens by the NCI. Anti-GD 2 antibody (Ab) ch14.18/CHO targets this antigen and effectively orchestrates innate immune effector mechanisms against GD 2 expressing malignancies. However, one on target effect of anti-GD 2 Abs is the induction of neuropathic pain when applied as short term infusion (STI) requiring co-medication with intravenous morphine. Here we investigated whether long-term infusion (LTI) of anti-GD 2 Ab ch14.18/CHO may have an improved pain toxicity profile and at the same time mediate an effective immune response in patients (pts) with high risk relapsed/refractory NB which translates to objective clinical responses and an improved survival. Methods: 97 pts received 6x10 6 IU/m 2 sc IL2 (d1-5; 8-12), LTI of 100 mg/m 2 ch14.18/CHO (d8-17) and 160 mg/m 2 oral 13-cis-RA (d19-32) in an ongoing SIOPEN Phase II study (APN311-202) (NCT01701479) (44 pts) and a closed single center program (53 pts) (APN311-303). Response assessments followed INRG criteria. Serum ch14.18/CHO concentration-time curves were determined by validated ELISA methods and pharmacokinetics parameter were analyzed using standard non-compartmental methods. Fcγ;-receptor polymorphisms FCGR2A (H131R), -3A (V158F) and -3B (NA1/NA2) and patient-specific antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and whole blood cytotoxicity (WBT) responses were determined. Results: LTI was associated with low pain scores and a decreasing morphine usage compared to STI. Clinical overall responses were 30% (APN311-303) and 31% (APN311-202). The survival update of the APN311-303 cohort revealed a 1-y & 4-y OS of 94.2±3.2% & 60.9±9.0% (median FU 2.9y [0.7-5.2y]) and a 1-y & 4-y PFS of 54.4±6.9% & 32.3%±6.9% (median FU 2.8y [0.7 - 4.9y]). Median TTP was 571d (95% CI: 232.7d). The comparator is the reported historical gold standard for relapsed refractory NB patients with 1-y & 4-y PFS of 19±2% & 8±3% and OS of 56±3% & 14±4% and a median TTP of 63 d (95% CI: 56.8d). NB pts with high affinity FCGR alleles and an increase in ADCC (cut off 15%) are associated with longer PFS and OS rates (p Parameters of immune modulation (CDC, and WBT) and PK of ch14.18/CHO were comparable between APN311-202 and -303 cohorts. PK of ch14.18/CHO was analyzed in cycle 1: Cmax=12.2±0.4μg/ml, t½=8.4±1.1 d, AUC=145.3± 5.8 μg*d/ml, Vd ss =9.3±0.5L/m 2 . A pro-inflammatory cytokine response (IL-2, IL-6, IL-8, IFNγ) translated into the expansion of effector NK- (3x) and T-cells (2x). We observed HACA in 17/97 pts (17.5%) of which only 9/97 (9.3%) were neutralizing with respect to the inhibition of CDC and WBT activity. In HACA negative patients, levels of ch14.18/CHO and functional parameters (CDC and WBT) analyzed before subsequent treatment cycles indicate persistent anti-NB activity for the entire treatment period. Conclusion: LTI of ch14.18/CHO has an improved pain toxicity profile and at the same time is active and effective in high-risk relapsed/refractory NB. Citation Format: Holger N. Lode, Dominique Valteau-Couanet, Alberto Garaventa, Juliet Gray, Victoria Castel, Isaac Yaniv, Nikolai Siebert, Christian Jensen, Stefanie Endres, Lena Pill, Christin Eger, Diana Seidel, Madlen Juttner, Silke Kietz, Karoline Ehlert, Evelyne Janzek, Carla Manzitti, Ina Muller, Hans Loibner, Ruth Ladenstein. Immunotherapy with ch14.18/CHO in combination with IL2 is active and effective in high-risk relapsed/refractory neuroblastoma patients. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A032.

A multidimensional impedance platform for the real-time analysis of single and combination drug pharmacology in patient-derived viable melanoma models

In todays development of anticancer drugs, there is an enormous demand for sensitive, non-invasive real-time screening technologies to identify pharmacodynamics/-kinetics of single and combined drugs with high precision. The combination of sophisticated drug sensitivity testing with advanced in vitro tumor models reflecting heterogeneous tumor behavior in vivo is needed to more reasonably predict therapeutic outcome in vivo. In this study, the benefits of our real-time, non-invasive multidimensional impedance platform over standard in vitro drug sensitivity assays were demonstrated quantitatively using an advanced melanoma model. Detailed pharmacological profiles of clinically established targeted therapeutics in single and combination treatment have been identified in patient tissue and isolated 2D/3D cell line cultures. Impedance spectroscopy revealed significant differences in tissue structure responsible for BRAF inhibitor pharmacokinetics in BRAFV600E tumor microfragments and cell lines. Remarkably, BRAF-/MEK inhibitor combination treatment of direct patient-derived tissue, but not melanoma cell lines, resulted in short-term antagonistic effects consistent with in vivo findings. In contrast, the clinically validated resistance delay and thus long-term synergy of targeted therapeutics in advanced melanoma models has been demonstrated using impedance technology. The results demonstrate limited clinical transferability of 2D/3D cancer cell line-based chemosensitivity data and underline the importance of in vivo-like direct patient-derived tissue for predictive drug studies. Our non-invasive and highly sensitive multidimensional impedance platform offers great potential for quantifying short- and long-term drug kinetics and synergies to identify the most effective drug combinations in advanced cancer models, thereby improving personalized drug development and treatment planning and ultimately, overall patient outcomes.